Autophagy plays a key role in cell growth, development, aging, death process, as well as in tumor development, neurodegenerative diseases and their prognosis. In autophagy research, the characterization of autophagosomes in cells is often required, and microtubule-associated protein 1 light chain 3 protein (LC3) is a well-known autophagosome marker. However, recent studies have suggested that fluorescent protein tagged LC3 (FP-LC3) puncta do not always represent the autophagic vesicles, and in some case it may be LC3-involved protein aggregates. Thus, it puzzles the biologists how to accurately distinguish between the two types of FP-LC3 structures in living cells in order to avoid the inappropriate conclusions simply drawn by counting the FP-LC3 puncta numbers.
The research group at Britton Chance Center for Biomedical Photonics in Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology (HUST), headed by professor Zhihong Zhang, using a two-color fluorescence recovery after photobleaching (FRAP) technique, found that after photobleaching, the fluorescence signal of the early phagophore marker (e.g. WIPI1 protein)recovered rapidly, while the recovery of LC3 is either very slow or not happened. In contrast, in protein aggregates, the LC3 exhibited rapid fluorescence recovery, while other inherent components showed a very slow recovery rate after laser irradiation. Based on the distinct dynamic properties of FP-LC3 in the two types of punctate structures, we established a real-time and in-situ two-color FRAP technique for identification of LC3 fluorescent puncta in autophagosomes and protein aggregates, thus providing a valuable approach for the accurate quantification of the autophagy. This research result was published in Autophagy 2013 (IF 7.453) (LC3 fluorescent puncta in autophagosomes or in protein aggregates can be distinguished by FRAP analysis in living cells, Autophagy. 9 (5): 756-769, 2013).
This work was supported by the National Basic Research Program of China (Grant No. 2011CB910401), Science Fund for Creative Research Group of China (Grant No.61121004), National Natural Science Foundation of China (Grant No. 81172153), and Specific International Scientific Cooperation of China (Grant No. 2010DFR30820).